一贯煎对H<sub>2</sub>O<sub>2</sub>诱导L02细胞DNA氧化损伤的修复作用机制研究

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摘要目的探讨一贯煎对过氧化氢（H2O2）诱导的人正常肝细胞（L02）DNA氧化损伤的修复机制。方法将L02细胞随机分为6组，分别为正常对照组、模型对照组、一贯煎小剂量组、一贯煎中剂量组、一贯煎大剂量组及阳性对照组，每组各5只。正常对照组及模型对照组均加入含有20%正常大鼠血清的培养基，一贯煎小剂量组、一贯煎中剂量组、一贯煎大剂量组及阳性对照组分别加入20%一贯煎小剂量含药血清、20%一贯煎中剂量含药血清、20%一贯煎大剂量含药血清以及含有20%正常大鼠血清且Vc浓度为0.1 mmol/L的培养基。孵育24 h后，除正常对照组外，其余各组加入浓度为0.6 mmol/L的H2O2处理1 h。通过CCK-8检测氧化损伤细胞的活力；酶联免疫吸附试验（ELISA）检测DNA氧化损伤的标志物8-羟基脱氧鸟苷（8-OHDG）含量；流式细胞术检测活性氧（ROS）含量和细胞周期变化；逆转录-聚合酶链反应（RT-PCR）检测细胞ATM、CHK2、Cdc25A、Cdc25C的mRNA表达情况。结果 L02细胞活力随着H2O2浓度增高及作用时间延长而降低（P ＜ 0.05）；与正常对照组比较，模型对照组细胞活力下降（P ＜ 0.05）；8-OHDG含量增加（P ＜ 0.05）；ROS平均荧光强度显著增加（P ＜ 0.05）；G1期细胞比例减少，S期、G2/M期细胞比例增加（P ＜ 0.05）；ATM、CHK2的mRNA表达上调，Cdc25A、Cdc25C的mRNA表达下调（P ＜ 0.05）。与模型对照组比较，一贯煎小剂量组、一贯煎中剂量组、一贯煎大剂量组及阳性对照组细胞活力提高（P ＜ 0.05）；8-OHDG含量减少（P ＜ 0.05）；ROS平均荧光强度降低（P ＜ 0.05）；G1期细胞比例增加，S期、G2/M期细胞比例减少（P ＜ 0.05）；ATM、CHK2的mRNA表达下调，Cdc25A、Cdc25C的mRNA表达上调（P ＜ 0.05）。结论一贯煎可能通过抑制ATM/CHK2/Cdc25A/Cdc25C通路影响细胞周期进而修复L02细胞DNA氧化损伤。

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	王阿美张睿刘文兰

关键词 ：一贯煎, L02细胞, 细胞周期, DNA氧化损伤修复

Abstract：Objective To investigate the repair mechanism of Yiguanjian Decoction in hydrogen peroxide (H2O2)-induced DNA oxidative damage of human normal liver cells (L02). Methods L02 cells were randomly divided into 6 groups, including normal control group, model control group, low dose Yiguanjian Decoction group, middle dose Yiguanjian Decoction group, high dose Yiguanjian Decoction group and positive control group, five mice in each group. The normal control group and the model control group were cultured with medium containing 20% normal rat serum, simultaneously, low dose Yiguanjian Decoction group, middle dose Yiguanjian Decoction group, high dose Yiguanjian Decoction group were respectively cultured with medium containing 20% low, middle, high dose Yiguanjian Decoction of serum, and positive control group was cultured with 20% normal rat serum containing 0.1 mmol/L Vc. After 24 hours, except the normal control group, the cells of other groups were treated with 0.6 mmol/L H2O2 for 1 hour. The cell viability was detected by CCK-8; the content of 8-hydroxydeoxyguanosine (8-OHDG) was determined by enzyme linked immunosorbent assay (ELISA); the reactive oxygen species (ROS) contents and cell cycle were tested by flow cytometry; the mRNA levels of ATM, CHK2, Cdc25A and Cdc25C were assessed by revers transcriptation-polymerase chain reaction (RT-PCR). Results L02 cell viability was decreased with increasing H2O2 concentration and prolonged action time. Compared with the normal control group, cell viability of the model control group was decreased (P ＜ 0.05); 8-OHDG contents were increased (P ＜ 0.05); the mean fluorescence intensity of ROS was increased significantly (P ＜ 0.05); the cell proportion of G1 phase was decreased, while the cell proportion of S phase and G2/M phase were increased (P ＜ 0.05); the mRNA levels of ATM and CHK2 were up-regulated, and Cdc25A and Cdc25C were down-regulated (P ＜ 0.05). Compared with the model control group, the cell viability of the low dose Yiguanjian Decoction group, middle dose Yiguanjian Decoction group, high dose Yiguanjian Decoction group and positive control group were increased (P ＜ 0.05); the 8-OHDG content were decreased (P ＜ 0.05); the mean fluorescence intensity of ROS were decreased (P ＜ 0.05); the cell proportion of G1 phase were increased, the cell proportion of S phase and G2/M phase were decreased (P ＜ 0.05); the mRNA levels of ATM and CHK2 were down-regulated, while the expression of Cdc25A and Cdc25C mRNA were up-regulated (P ＜ 0.05). Conclusion Yiguanjian Decoction could repair the DNA oxidative damage of L02 cells by inhibiting the cell cycle through the ATM/CHK2/Cdc25A/Cdc25C pathway.

Key words： Yiguanjian Decoction L02 cells Cell cycle DNA oxidative damage

基金资助:北京市自然科学基金项目（7182021）。

通讯作者: 刘文兰（1968.5-），女，博士，教授，博士生导师；研究方向：慢性肝损伤中医证候病理基础研究。

作者简介: 王阿美（1990.9-），女，首都医科大学中医药学院2017级中医诊断学专业在读硕士研究生；研究方向：慢性肝损伤中医证候病理基础研究。

引用本文:

王阿美张睿刘文兰. 一贯煎对H₂O₂诱导L02细胞DNA氧化损伤的修复作用机制研究[J]. 中国医药导报, 2020, 17(2): 9-14,24.
WANG Amei ZHANG Rui LIU Wenlan. Study on the repair mechanism of Yiguanjian Decoction in DNA oxidative damage of L02 cells induced by H₂O₂. 中国医药导报, 2020, 17(2): 9-14,24.

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